Several hundreds of proteins/peptides are known to cause membrane damage and to lead to cell death. They are found in many organisms such as plants, insects, frogs, snakes, and humans. Many such peptides are used by nature as either host defense (antimicrobial peptides) or offense (toxins from spider and snake venom). Since the body produces peptides that act against its own cells, the cytotoxic peptides are involved in “autocytotoxic” conditions. In addition to the wild-type cytolytic peptides, synthetic peptides modified from naturally occurring cytolytic peptides can also be cytotoxic, with mutation-sensitive activities. The size of the protein ranges from a short peptide to several hundreds of amino acids. Both large proteins and small peptides share a common feature, the ability to create a leakage pathway for molecules and ions to cross lipid bilayers.
The monomeric structures of small cytolytic peptides can be classified into three groups: α-helix, β-sheet, and loop-strand. These peptides adopt totally different structures under different conditions, allowing the conformation to adjust to the surrounding environment. Most α-helical cytolytic peptides are largely random coil in solution and change to an α-helix within the membrane. Theft-strand and loop-strand peptides often have disulfide S—S bond(s) that constrain the peptide conformation. Even though it is uncertain whether these disulfide bond-constrained peptides will have similar conformations in solution and in the membrane, it is commonly assumed that the disulfide bonds will keep the β-sheet structure unchanged. The most prominent characteristic of the small cytolytic peptides is that they are largely amphipathic, with hydrophobic and positively charged hydrophilic regions (with an excess of Lys and Arg residues). Positively charged residues can facilitate an initial contact between the peptides and the polyanionic sites in the membrane. Amphipathicity is essential for the membrane disruption effects by these peptides.
One of the best characterized β-sheet cytolytic peptide is protegrin. Native protegrins were originally purified from porcine leukocytes. There are five known porcine protegrins, PG-1-PG-5. These peptides share a common feature and conformation. Protegrin (PG-1) forms the f-hairpin conformation, which is composed of 18 amino acids (RGGRL-CYCRR-RFCVC-VGR) with a high content of cysteine (Cys) and positively charged arginine (Arg) residues. Formation of two disulfide bonds between the cysteine residues in PG-1 is crucial for the peptide activity, since the activity can be restored by stabilizing the peptide structure. It was noted that the translocation ability of PG-1 is coupled to its pore-formation capacity and depends on its folding to the β-hairpin conformation. PG-1 is a very potent antibiotic peptide, and experimental NMR study has revealed the three-dimensional structure of PG-1 in solution.
The interaction of protegrin with the membrane strongly depends on its lipid composition. Recent experimental studies have shown that PG-1 inserts readily into a membrane composed of negatively charged anionic lipids with the phosphatidylglycerol (PG) headgroup but significantly less into a membrane composed of neutrally charged lipids with the phosphatidylcholine (PC) or phosphatidylethanolamine (PE) headgroups. The major role of protegrins in the membrane is a channel formation that causes an ion leakage. Recently, it has been reported that either PG-1 or PG-3 can induce weak anion-selective channels and potassium leakage from liposomes and that PG-3 formed moderately cation-selective channels in the presence of bacterial lipopolysaccharide in planar phospholipid bilayers. Formation of protegrin channels disrupts the membrane surface by creating a pore across the membrane. PG-1 adopts a dimeric structure in DPC (dodecylphosphocholine) micelles, and a channel is formed by the association of several dimmers.
Normally, peptides suffer from a short in vivo half life, sometimes mere minutes, making them generally impractical, in their native form, for protegrin administration. Thus there exists a need in the art for modified protegrin peptides having an enhanced half-life and/or reduced clearance as well as additional protegrin advantages as compared to the protegrin peptides in their unmodified form.